1. Field of the Invention
The present invention relates to synthetic peptides capable of eliciting fibrin-specific antibodies free of fibrinogen cross-reactivity. This invention also relates to hybridoma cell lines secreting antifibrin-specific monoclonal antibodies free of cross-reactivity with fibrinogen, the circulating fibrin precursor. These antibodies have been found to be useful as agents for the in vivo localization of fibrin deposits and the detection of thrombi in humans and other animals.
2. Description of the Background Art
In vivo immunodetection of thrombi and fibrin deposits remains an important clinical problem. In humans, the detection and localization of deep vein thrombi and coronary artery thrombi are two clinically important problems.
Blood clots when thrombin cleaves two pairs of small peptides from fibrinogen to yield fibrin monomers (Blomback, B. and Vestermark, A., Ark. Kemi 12: 173 (1958) and Doolittle, R. F., Adv. Protein Chem. 27: 1 (1973)). Fibrin monomers spontaneously aggregate to form an insoluble gel, which is then covalently stabilized by Factor XIIIa. Despite the dramatic differences between the two, fibrin retains 98% of the original covalent structure of fibrinogen. Thus, it is understandable that antifibrin serum cross-reacts strongly with fibrinogen, and that only one instance is known where a fibrin-specific serum was produced (Bosnjakovic, V. B. et al., Lancet 2: 452 (1977)).
Polyclonal antibodies raised against fibrin or fibrinogen have been utilized for localizing a variety of tumors, in particular the fast-growing kind (Day, E. D., et al., J. Natl. Cancer Insti. 22: 413 (1958), Bale, W. F., et al., Cancer Res. 20: 1488 (1960), Pressman, D., Cancer Res. 40: 2965 (1980)). However, these antibodies all showed to some extent cross-reactivity with fibrinogen.
Polyclonal antibodies to fibrin-fibrinogen, to fibrinogen alone or to degradation products of both polypeptides have been developed for use in the detection of venous thrombosis in humans (Reich, T., et al., Surgery 60(6): 1211, Spar, I. L., et al., Circulation Research, XVII: 322 (1965), and Stephan, W., et al., U.S. Pat. No. 4,147,765). In Reich, T., et al. and Spar, I. L., et al., supra, the radiolabelled antibodies had a substantial degree of fibrin-fibrinogen cross-reactivity. In the Stephan, W., et al. reference, the antiserum was elicited by immunological challenge with the early fibrin and fibrinogen degradation products formed by the action of plasmin on fibrin and fibrinogen, and referred to in the literature as fibrinogen S (fg-X), and fibrinogen Y (fg-Y).
A different approach for the detection of deep-vein thrombosis was undertaken by Knight, L. C., et al., J. Nuclear Med. 19(8): 891 (1978) by radio-iodinating fibrinogen. This method, however, was found to be inferior to the labelling of platelets with .sup.111 In, for the localization of thrombi in deep-vein thrombosis.
When plasminogen is converted by an activator to plasmin, the active fibrinolytic enzyme of plasma, it develops a marked affinity for its substrate, fibrin. Ouchi, H. and Warren, R., Surgery 51(1): 42 (1962), discovered that this affinity might be put to use by tagging plasminogen with a radioactive isotope and using it as a tracer to seek out intravascular clots. However, the .sup.131 I-labelled plasminogen showed a somewhat depressed fibrinolytic activity (28.6%).
Pilgeram, L., U.S. Pat. No. 4,245,040 improved on an earlier method for detecting circulating fibrin (Lorand, L., in Fibrinogen and Fibrin Turnover of Clotting Factors, Ed. Koller F., F. K. Schattauer Verlag-Stuttgart, 1963; and Kisker, C. D. and Rush, R., in "Detection of Intravascular Clotting," J. Clin. Investig. 50:2235 (1971)). Pilgeram's improvement consisted in attaching an isotopic label to the soluble circulating fibrin, by means of a Factor XIII-containing glycine-C.sup.14 ethyl ester. However, it is recognized that the use of antifibrin antibodies which crossreact with fibrinogen is limited.
Although not previously done in the case of the fibrin molecule, synthetic peptides corresponding to portions characteristic of certain antigens have been prepared. For example peptides corresponding to carcinoembrionic antigen (CEA) are known in the prior art, and have been used for raising antibodies (Sela, M., et al., U.S. Pat. No. 4,075,194). The thus raised antibodies were intended for use in the detection of adenocarcinomas of the digestive tract, pancreas and breast.
A need continues to exist for highly specific antifibrin monoclonal antibodies and for synthetic epitopic peptides capable of raising such nonfibrinogen-cross-reacting antibodies. The antibodies would be useful for the in vivo detection of thrombi and fibrin deposits.